Method for detecting mitochondria gene alterations

ABSTRACT

The present invention relates to a method for detecting mitochondria alterations, which comprises the following steps: (A) providing a separation element and a sample; (B) mixing the separation element and the sample, wherein a detecting sample is obtained through the binding of a DNA fragment on the separation element to mitochondrial DNA in the sample; (C) dividing the detecting sample into a comparison group and a detection group; (D) adding an amplification solution into the comparison group and the detection group respectively to begin a DNA amplified reaction, and further adding a restriction enzyme into the detection group, wherein the amplification solution comprises a labeling reagent and a primer pair; and (E) detecting amounts of the labeling reagent in the comparison group and the detection group respectively after the DNA amplified reaction.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting mitochondriagene alterations and, more particularly, to a method for detectingmitochondria gene alterations by means of a single primer pair.

2. Description of Related Art

Mitochondria are the main sites in cells where oxidative phosphorylationoccurs and adenosine triphosphate is synthesized, and energy to humanand animal cells comes from. Furthermore, mitochondria not only canprovide energy, they also work to take part in apoptosis, cellulardifferentiation and signaling, as well as cell growth and cell cycle.Hence, whether the mitochondria genes are defective or not isinstrumental to the functioning of human and animal cells.

Recent studies have found that mitochondria genes can be madesusceptible to gene variations, including mutations and deletions, whenexposed to attack of free radicals that are generated during oxidation.Such alterations in mitochondria genes can heavily influence biologicalfunctions related to mitochondria or can even lead to cell death. Anexample of known diseases or symptoms incurred by mitochondria genealterations includes degenerative diseases. Some studies have alsosuggested that link between mitochondria gene alterations and cancers.Hence, detection for alterations in mitochondria genes would prove to behelpful for diagnosing diseases in clinical practices.

The majority of currently understood methods for detecting mitochondriagene alterations are focused on using direct DNA sequencing; however,the technicality of direct DNA sequencing is complicated and thematerial and the device required in its operation are expensive, so thistechnique has not been widely received by testing services andinstitutions.

In response, it is desirable to provide a rapid and simple device andmethod for detecting mitochondria gene alterations, which can be appliedto clinical uses to examine mitochondria gene related diseases, and fromwhich to use as a reference for treating or diagnosing diseases.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for detectingmitochondria gene (mtDNA) alterations, which can not only determineoccurrence of mitochondria gene alterations, but also quantify the same.

Another object of the present invention is to provide a device and asystem for detecting mtDNA alterations, wherein the device has a threedimensional structure. Hence, extraction and detection processes can beperformed on the mitochondria genes in a single device, and making itpossible to determine in a rapid and precise fashion whether there aregene alterations in the mitochondria genes. On a particular note, thedevice and the system of the present invention are the resulting deviceand system realized from using the present invention's method fordetecting mtDNA alterations.

To achieve the above-identified object, the device of the presentinvention comprises: a purification unit containing a first reactionchamber and a separation-element disposed chamber, wherein theseparation-element storage chamber connects to the first reactionchamber through a first pump; and a detection unit disposed under thepurification unit, wherein the detection unit comprises: a secondreaction chamber and a detecting-sample chamber, the second reactionchamber connects to the first reaction chamber, and the detecting-samplechamber connects to the second reaction chamber through a second pump.Herein, the first reaction chamber and the second reaction chamberconnect to each other. More specifically, the first reaction chamber andthe second reaction chamber connect to each other to form a reactionchamber.

In the aforementioned device of the present invention, when a samplecontained with mitochondria genes is placed into the first reactionchamber, a separation element contained in the separation-elementstorage chamber can be introduced into the first reaction chamber by thefirst pump. Then, the separation element is mixed with the sample toperform an extraction process on the mitochondria genes. The firstreaction chamber and the second reaction chamber connect to each other,so the sample contained in the reaction chamber can be later introducedinto the detecting-sample chamber by the second pump after theextraction process and then a process for detecting mitochondria genealterations is performed on the sample introduced into thedetecting-sample chamber. Then, due to the interconnection between thefirst reaction chamber and the second reaction chamber, thedetecting-sample obtained from the extraction process of the separationunit can be introduced into the detecting-sample chamber through thehelp of the second pump for carrying out detection for mitochondriagenes alterations and the like.

According to the aforementioned device of the present invention, theextraction and detection processes can be performed on the mitochondriagenes in a single device. Hence, in contrast to the case with aconventional device that the extraction process has to be performed inconjunction with another extraction device, the whole series ofdetection steps can be further simplified by using the device of thepresent invention.

In the device for detecting mtDNA alterations of the present invention,the purification unit may further comprises a washing-solution disposedcamber, which connects to the first reaction chamber through a thirdpump. When the separation element contained in the separation-elementstorage chamber is mixed with the sample, a washing solution containedin the washing-solution storage chamber can be introduced into the firstreaction chamber by the third pump to remove other matrix in cellsexcept for the mitochondria genes. Herein, the washing solution is notparticularly limited, and can be any cell-washing solution generallyused in the art, such as PBS.

In addition, the device for detecting mtDNA alterations of the presentinvention may further comprise a micro-mixture unit connecting to thefirst reaction chamber in order to extract and wash the sample moreuniformly.

In one aspect of the device for detecting mtDNA alterations of thepresent invention, the detection unit may further comprise: a comparisonchamber connecting to the second reaction chamber through the secondpump. Herein, DNA amplified reactions can be selectively performed onthe samples contained in the comparison chamber and the detecting-samplechamber. Then, DNA signals emitted from the comparison chamber arecompared with those emitted from the detecting-sample chamber todetermine whether the mitochondria genes are alternated or not.

In another aspect of the device for detecting mtDNA alterations of thepresent invention, the detection unit may further comprise: amitochondria-gene checking chamber and a temporary chamber, wherein themitochondria-gene checking chamber and the temporary chamber connect tothe second reaction chamber through a fourth pump, and the temporarychamber also connects to the detecting-sample chamber through the secondpump. Herein, the DNA amplified reaction can be firstly performed on thesample contained in the mitochondria-gene checking chamber to determinewhether the mitochondria genes are extracted by the separation elementor not. After the checking process confirmed that the extraction processis successful, the sample contained in the temporary chamber is thenintroduced into the detecting-sample chamber through the second pump toperform the sequential detection process.

In the aforementioned aspect of the device for detecting mtDNAalterations, the detection unit may further comprise: a comparisonsample chamber, which connects to the temporary chamber through thesecond pump. Hence, the sample contained in the temporary chamber can beintroduced into the detecting-sample chamber and the comparison samplechamber by the second pump, and then DNA amplified reactions and otherreactions (such as restriction enzyme digestion) can be selectivelyperformed on the samples contained in the detecting-sample chamber andthe comparison sample chamber. Finally, the signals emitted from thecomparison sample chamber are compared with those emitted from thedetecting-sample chamber to determine whether the mitochondria genes arealternated or not.

The devices for detecting mtDNA alterations according to all theaforementioned aspects of the present invention may further compriseplural gas inlets, wherein each gas inlets connects to each elements ofthe purification unit or the detection unit such as the first pump, thesecond pump, the third pump, the forth pump, the micro-mixture unit, andthe temporary chamber by corresponding connection paths.

In addition, in the devices for detecting mtDNA alterations according toall the aforementioned aspects of the present invention, the first pump,the second pump, the third pump and the forth pump can be any micro-pumpgenerally used in the art such as peristaltic micropumps andsuction-type micropumps. Preferably, the first pump and the third pumpare peristaltic micropumps respectively, and the second pump and theforth pump are suction-type micropumps respectively.

Furthermore, in the devices for detecting mtDNA alterations according toall the aforementioned aspects of the present invention, all theelements are in micro-sized, so the length, the weight and the height ofthe whole devices can be confined to several tens of millimeters (mm).Hence, the devices of the present invention can be designed as portableand disposable devices.

Except for the aforementioned devices for detecting mtDNA alterations ofthe present invention, the present invention further provides a systemusing the aforementioned devices. The system for detecting mtDNAalterations of the present invention comprises: the aforementioneddevice for detecting mtDNA alterations; a temperature controllerdisposed surrounding the periphery of the device to control atemperature of the device; and an analysis device disposed over thedevice to detect signals emitting from the device.

In the system of the present invention, the temperature controller isused to control the temperature of the device, in order to perform thedetection process such as the DNA amplified reaction. Herein, thetemperature controller may comprise: a sensing module and a controlmodule. The sensing module can detect the temperature of the device, andthen the temperature of the device is increased or decreased by thecontrol module.

In addition, the system for detecting mtDNA alterations of the presentinvention may further comprise: a heater/cooler device, which isdisposed under the device for detecting mtDNA alterations and connectsto the temperature controller. The heater/cooler device can change thetemperature of the device rapidly. In the present invention, theheater/cooler device is not particularly limited, and can be anyheater/cooler device generally used in the art, such as a thermoelectriccooler (TE cooler), a hot plate and a MEMS heater. Preferably, theheater/cooler device used in the present invention is a TE cooler. It isbecause that the TE cooler can increase and decrease temperaturerapidly. In addition, the TE cooler further can increase and decreasetemperature in large area, and the temperature range thereof is broad(−10° C. to 200° C.).

Furthermore, in the system for detecting mtDNA alterations of thepresent invention, the analysis device can be selected according to theDNA signal to be detected. The analysis device used in the presentinvention can be any analysis device generally used in the art, such asan electrophoresis device, a fluorescence device and a UV-lightdetecting device. Preferably, the analysis device used in the system ofthe present invention is a fluorescence device. More preferably, theanalysis device used in the system of the present invention is afluorescence device equipped with a photomultiplier tube (PMT).

When magnetic beads are used to extract the mitochondria genes in thedevice for detecting mtDNA alterations of the present invention, thesystem for detecting mtDNA alterations of the present invention mayfurther comprise an electromagnetic controller, which provides amagnetic field to the device for detecting mtDNA alterations. Hence,when the electromagnetic controller provides a magnetic field to thedevice, the magnetic beads can be absorbed by the magnetic field. Inthis case, it is unnecessary to perform an additional separation step toobtain the purpose of extracting the mitochondria genes.

Except for the aforementioned device and system for detecting mtDNAalterations, the present invention further provides a method fordetecting mtDNA alterations, and this method is particularly suitablefor the device and the system of the present invention. However, themethod of the present invention is not limited to be applied to thedevice and the system of the present invention, and it can also beapplied to other devices and systems.

The method for detection mtDNA alterations of the present inventioncomprises the following steps: (A) providing a separation element and asample contained with mitochondria genes, wherein the separation elementis modified with a DNA fragment for recognizing mitochondria genes; (B)mixing the sample and the separation element to separate themitochondria genes from the sample through a binding between the DNAfragment of the separation element and the mitochondria genes in thesample, and sequentially obtaining a detecting sample contained with themitochondria genes; (C) dividing the detecting sample into a comparisongroup and a detection group; (D) adding an amplification solution intoboth the comparison part and the detection part respectively and furtheradding a restriction enzyme into the detection group to perform a DNAamplified reaction, wherein the amplification solution comprises alabeling reagent and a primer pair; and (E) detecting amounts of thelabeling reagent in the comparison group and the detection grouprespectively after the DNA amplified reaction, wherein when the amountof the labeling reagent in the comparison part is different from that inthe detection group, it indicates that a mtDNA alteration is present inthe sample.

It should be noted that the primer pairs added into the comparison groupand the detection group are the same primer pairs. Hence, when themethod of the present invention is used, the purpose of detecting mtDNAalterations can also be accomplished at the expense of only a singleprimer pair. However, when the conventional method is used, two primerpairs has to be used, wherein one primer pair is paired with normalsequence of a target gene region, and the other primer pair is pairedwith mutated sequence of the target gene region. Hence, the method ofthe present invention is more convenient than the conventional method.

More specifically, in the method of the present invention, when theamount of the labeling reagent in the detection group is less than thatin the comparison group, it indicates that a mtDNA alteration is presentin the sample.

In addition, the restriction enzyme used in the step (D) can be arestriction enzyme capable of digesting DNAs in a target gene region ofthe mitochondria genes, and the target gene region can be a mutationregion to be detected in the mitochondria genes. Herein, the restrictionenzyme is selected according to the sequence of the target gene regionin the mitochondria genes, and can be any restriction enzyme generallyused in the art.

In the method for detection mtDNA alterations of all aspects of thepresent invention, the primer pair used in the step (D) is a primer pairto amplify a target gene region of the mitochondria genes. Herein, anyDNA amplified reaction such as a polymerase chain reaction (PCR) and areal-time polymerase chain reaction (real-time PCR) can be used in themethod of the present invention to amplify the mitochondria genes.

In addition, the labeling reagent used in the step (D) can be a dyedetected with fluorescence or UV-light. Preferably, the labeling reagentis a fluorescent dye. More preferably, the labeling reagent is afluorescent dye such as SybrGreen that can emit fluorescence when itchelates into major grooves of DNAs.

The methods for detection mtDNA alterations of all aspects of thepresent invention may further comprise a step (B′) after the step (B):dividing the detecting sample into a mitochondria gene-checking groupand a detecting sample group, and detecting whether the mitochondriagenes are present in the mitochondria gene-checking group or not. Inthis case, the step (C) is: dividing the detecting sample group into acomparison group and a detection group. Only when it is confirmed thatthe mitochondria genes are present in the mitochondria gene-checkinggroup, the sequential steps (C) and (D) are performed. In the step (B′),any conventional method generally used in the art such as a PCR and areal-time PCR can be used to check whether the mitochondria genes arepresent in the mitochondria gene-checking group or not. When the PCR orthe real-time PCR are used to check the mitochondria genes in themitochondria gene-checking group, the used primer pair is notparticularly limited, as long as it can amplify the mitochondria gene.Preferably, the used primer pair can pair with a D-loop region of themitochondria genes.

In the method for detecting mtDNA alterations of all aspects of thepresent invention, the separation element used in the step (A) can beany separation element generally used in the art, such as a magneticbead or a polymer bead. When the magnetic bead is used as the separationelement, the mitochondria genes can be extracted with a magnetic field.When the polymer bead is used as the separation unit, the extraction ofthe mitochondria gene can be accomplished by gravitational orcentrifugal force.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view showing a method for detecting mtDNAalterations according to Embodiment 1 of the present invention;

FIG. 2A is a exploded view showing a device for detecting mtDNAalterations according to Embodiment 1 of the present invention;

FIG. 2B is a perspective view showing a device for detecting mtDNAalterations according to Embodiment 1 of the present invention;

FIG. 3 is a perspective view showing a system detecting mtDNAalterations according to Embodiment 1 of the present invention;

FIG. 4 is a result of a checking step of an extraction process onmitochondria genes according to Example 1 of the present invention;

FIG. 5 is a detecting result of normal mtDNA group according to Example1 of the present invention;

FIG. 6 is a detecting result of mutated mtDNA group according to Example1 of the present invention;

FIG. 7 is a detecting result of normal mtDNA group according toComparative Example 1 of the present invention;

FIG. 8 is a detecting result of mutated mtDNA group according toComparative Embodiment 1 of the present invention; and

FIG. 9 is a detecting result according to Example 2 and ComparativeExample 2 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

In the following embodiments of the present invention, the figures aresimplified perspective views. However, only the elements relate to thepresent invention are shown in these figures. These shown embodimentsare not actual performance aspects. The numbers, the shapes and thesizes of the shown elements are only one selective design, and they maybe more complicated.

Embodiment 1

FIG. 1 is a perspective view showing a method for detecting mtDNAalterations of the present embodiment, FIG. 2A and FIG. 2B arerespectively a exploded view and a perspective view showing a device fordetecting mtDNA alterations of the present embodiment, and FIG. 3 is aperspective view showing a system detecting mtDNA alterations of thepresent embodiment.

As shown in FIG. 2A and FIG. 2B, the device for detecting mtDNAalterations of the present embodiment comprises: a purification unit 21containing a first reaction chamber 211 and a separation-element storagechamber 212, wherein the separation-element storage chamber 212 connectsto the first reaction chamber 211 through a first pump 214; and adetection unit 22 disposed under the purification unit 21, wherein thedetection unit 22 comprises: a second reaction chamber 221 and adetecting-sample chamber 224, the second reaction chamber 221 connectsto the first reaction chamber 211, and the detecting-sample chamber 224connects to the second reaction chamber 221 through a second pump 228.Herein, the first pump 214 is a peristaltic micropump, and the secondpump 228 is a suction-type micropump. In addition, a connection path 217is disposed between the first pump 214 and the separation-elementstorage chamber 212, and a separation element contained in theseparation-element storage chamber 212 is introduced into theseparation-element storage chamber 212 by the first pump 214 to reactwith a sample contained in the first reaction chamber 211.

In addition, in the device for detecting mtDNA alterations of thepresent embodiment, the purification unit 21 further comprises awashing-solution storage chamber 213, which connects to the firstreaction chamber 211 through a third pump 215. In addition, a connectionpath 217 is disposed between the third pump 215 and the washing-solutionstorage chamber 213, and a washing solution (such as water) contained inthe washing-solution storage chamber 213 is passed through theconnection path 217 and introduced into the first reaction chamber 211by the third pump 215. Herein, the third pump 215 is a peristalticmicropump.

In the device for detecting mtDNA alterations of the present embodiment,the detection unit 22 further comprises: a mitochondria-gene checkingchamber 223 and a temporary chamber 222, wherein the mitochondria-genechecking chamber 223 and the temporary chamber 222 connect to the secondreaction chamber 221 through a fourth pump 226, and the temporarychamber 222 also connects to the detecting-sample chamber 224 throughthe second pump 228. Herein, connection paths between themitochondria-gene checking chamber 223/the temporary chamber 222 and thefourth pump 226 are back pressure paths 227, and the sample contained inthe second reaction chamber 221 can be divided equally into themitochondria-gene checking chamber 223 and the temporary chamber 222. Inaddition, connection paths between the temporary chamber 222 and thesecond pump 228 and between the second reaction chamber 221 and thefourth pump 226 are back pressure paths.

Furthermore, in the device for detecting mtDNA alterations of thepresent embodiment, the detection unit 22 further comprises: acomparison sample chamber 225, which connects to the temporary chamber222 through the second pump 228. Herein, connection paths between thecomparison sample chamber 225/the detecting-sample chamber 224 and thesecond pump 228 are back pressure paths, and the sample contained in thetemporary chamber 222 can be divided equally into the comparison samplechamber 225 and the detecting-sample chamber 224 to perform thesequential detection process.

In addition, in the device for detecting mtDNA alterations of thepresent embodiment, the detection unit 22 further comprises: plural gasinlets 23, wherein each gas inlets 23 respectively connects to the firstpump 214, the second pump 228, the third pump 215, the forth pump 226,the micro-mixture unit 216, the connection path between the secondreaction chamber 221 and the fourth pump 226, the connection pathbetween the mitochondria-gene checking chamber 223 and the temporarychamber 222, the connection path between the temporary chamber 222 andthe second pump 228, and the connection path between thedetecting-sample chamber 224 and the comparison sample chamber 225.Compressed gas can be introduced from the gas inlets 23 to facilitatethe sample flowing between each connection path.

Then, as shown in FIG. 3, the system for detecting mtDNA alterations ofthe present embodiment comprises: the aforementioned device 2 fordetecting mtDNA alterations; a temperature controller 4 disposedsurrounding the device 2 to control a temperature of the device 2,wherein the temperature controller 4 is disposed under the device 2 inthe present embodiment; and an analysis device 5 disposed over thedevice 2 to detect signals emitting from the sample contained in thedevice 2. Herein, the system for detecting mtDNA alterations of thepresent embodiment further comprises a heater/cooler device 3, which isdisposed under the device 2 and connects to the temperature controller4. In the present embodiment, the heater/cooler device 3 is a TE cooler.

In addition, the temperature controller 4 of the present embodimentcomprises: a temperature sensing module 41, a power supply 42, and acontrol module 43. The temperature sensing module 41 can detect thetemperature of the device 2. The control module 43 can control the powersupply 42 providing power to the heater/cooler device 3 based on thetemperature detected by the temperature sensing module 41 to increase ordecrease the temperature of the device 2. Herein, the detailed structureof the temperature controller 4 is only one embodiment of the presentinvention, and the present invention is not limited thereto.

Furthermore, the analysis device 5 of the present invention is afluorescence device, which comprises: a processor 51 and aphotomultiplier tube 52. The photomultiplier tube 52 provides anexcitation light to the device 2 and receives signals emitted from thedevice 2, and the signals received by the photomultiplier tube 52 areoutputted by the processor 51. The photomultiplier tube 52 can convertphoto signals into electric signals, and amplify the electric signals toobtain the signals emitted from the device 2. Herein, the detailedstructure of the analysis device 5 is only one embodiment of the presentinvention, and the present invention is not limited thereto.

The system for detecting mtDNA alterations of the present embodimentfurther comprises an electromagnetic controller 6, which provides amagnetic field to the device 2. Herein, the electromagnetic controller 6comprises: a power supply 61, a vacuum pump 62, a digital controller 63,and a solenoid valve 64. The power supply 61 provides power to thevacuum pump 62 and the digital controller 63, and then signals aretransmitted to the solenoid valve 64 to output a magnetic field to thedevice 2. Herein, the detailed structure of the electromagneticcontroller 6 is only one embodiment of the present invention, and thepresent invention is not limited thereto.

Furthermore, the system for detecting mtDNA alterations of the presentembodiment further comprises an object lens 7. The excitation lightemitted from the analysis device 5 can be focused on a detection regionby the object lens 7, and then the object lens 7 can receive signalsemitted from the excited sample to improve the detection effect of theanalysis device 5.

Hereafter, the device, the system and the method for detecting mtDNAalterations of the present embodiment are explained accompanied withFIG. 1 to FIG. 3 of the present invention.

First, as shown in FIG. 1, FIG. 2A and FIG. 3, a sample 11 (i.e. cells)contained with mitochondria genes is provided in the first reactionchamber 211 (as shown in FIG. 1(a)), and a separation element 12 (suchas magnetic beads) is provided in the separation-element disposedchamber 212 of the device 2. After the sample 11 is lysed, theseparation element 12 contained in the separation-element disposedchamber 212 is introduced into the first reaction chamber 211 throughthe first pump 214 (as shown in FIG. 1(b)), and then the sample 11 iswell mixed with the separation element 12 by the micro-mixture unit 216.Mitochondria genes 111 can bind to the separation element 12 via a DNAfragment for recognizing mitochondria genes modified on the separationelement 12. The electromagnetic controller 6 provides a magnetic fieldto the device 2 to separate the mitochondria genes 111 from other matrixof the cells. Then, a washing solution contained in the washing-solutionstorage chamber 213 is introduced into the washing-solution storagechamber 213 to remove other matrix of the cells (as shown in FIG. 1(c))to obtain a detecting sample.

Next, as shown in FIG. 1, FIG. 2B and FIG. 3, the first reaction chamber211 and the second reaction chamber 221 together form a reactionchamber, the fourth pump 226 can introduce the detecting samplecontained in the second reaction chamber 221 into the mitochondria-genechecking chamber 223 and the temporary chamber 222, and the detectingsample is divided into a mitochondria gene-checking group and adetecting sample part (as shown in FIG. 1(d) and (e)). Then, anamplification solution is added into the mitochondria-gene checkingchamber 223 to identify whether the mitochondria genes are present ornot. This step is a checking step of an extraction process onmitochondria gene. The amplification solution comprises a labelingreagent and a primer pair. The labeling reagent can be any DNA labelingreagent generally used in the art, and the sequence of the primer paircan be any sequence selected from the mitochondria gene. In the presentembodiment, the labeling reagent is a reagent which can chelate intomajor grooves of DNAs to emit fluorescence, and the sequence of theprimer pair contains a sequence that can pair with a D-loop region ofthe mitochondria genes. After a PCR or a real-time PCR is performedtogether with a heater/cooler device 3 to amplify DNAs, that is, to formamplimers 13, which are contained in the detecting sample, a lightsource 53 provides light on the detecting sample, and whether themitochondria genes are present in the mitochondria gene-checking groupof the detecting sample or not is determined by the photomultiplier tube52 of the analysis device (as shown in FIG. 1(d)).

As shown in FIG. 1, FIG. 2A and FIG. 3, when it is confirmed that themitochondria genes are indeed present in the mitochondria gene-checkinggroup of the detecting sample, the detecting sample group of thedetecting sample contained in the temporary chamber 222 is divided intoa comparison group (as shown in FIG. 1(f)) in the comparison samplechamber 225 and a detection group (as shown in FIG. 1(h)) in thedetecting-sample chamber 224 by the second pump 228 to perform adetection process. An amplification solution is added into thecomparison group in the comparison sample chamber 225 and the detectiongroup in the detecting-sample chamber 224 respectively, a restrictionenzyme is also added into the detection group in the detecting-samplechamber 224, and then a DNA amplified reaction is performed. Theamplification solution comprises a labeling reagent and a primer pair.The labeling reagent can be any DNA labeling reagent generally used inthe art, and the sequence of the primer pair can be a target gene regionselected from the mitochondria genes. In the present embodiment, thelabeling reagent is a reagent which can chelate into major grooves ofDNAs to emit fluorescence, the sequence of the primer pair contains asequence that can pair with a target gene region selected from themitochondria genes, and the restriction enzyme is an enzyme capable ofdigesting DNAs in the target gene of the mitochondria genes. It shouldbe noted that the primer pair added into the comparison group is thesame as that added into the detection group. After a PCR or a real-timePCR is performed together with a heater/cooler device 3 to amplify DNAscontained in the detecting sample, the light source 53 provides light onthe detecting sample, and whether the mitochondria genes in thedetecting sample are mutated or not is determined by the photomultipliertube 52 of the analysis device (as shown in FIG. 1(g) and (i)). When theamount of the labeling reagent in the detection group is less than thatin the comparison group by analyzing with the analysis device 5, itindicates that a mitochondria mutation is present in the sample.

EXAMPLE 1

The device, the system and the method for detecting mtDNA alterationsaccording to Embodiment 1 are used in the present example. The device,the system and the method used in the present example are the same asthose in Embodiment 1, so the detailed description is omitted herein.

Mutated Mitochondria Gene (mtDNA) Group

Magnetic beads (1.08 μm, Dynabeads® MyOne™ Carboxylic Acid, Invetrogen,USA) surface-immobilized with specific DNA fragments were used, whereinthe sequence of the DNA fragments was TGGTATTTTCGTCTGGGGGGTATG (SEQ IDNO: 1); the washing solution was de-ionized water (DI); the cell lineswere Lu03 (cell lines with A3243G point-mutated mtDNA); the primer pairof the amplification solution added into the mitochondria-gene checkingchamber had sequences shown in the following Table 1 (as shown in SEQ IDNOs: 4 and 5); the used restriction enzyme is ApaI, which can digest thesequence with A3243G point-mutated mtDNA but not digest normal mtDNAwithout mutations); the DNA amplified reaction was a PCR; the analysisdevice comprised a PMT system (C3830, R928; Hamamatsu Photonics, Japan);eight electromagnetic valves (EMVs; S070M-5BG-32, SMC, Japan) were used;the heater/cooler device was a TE cooler, wherein the workingtemperatures were set 60° C. for cell lysis, 37° C. for enzyme digestionand 95° C., 58° C. and 72° C. for PCR amplification; the labellingreagent was a fluorescent reagent contained in KAPA SYBR® FAST qPCR kits(KK4603, Kapa Biosystems, MA, USA); and the target region of themitochondria genes was A3243G.

Normal mtDNA Group

The conditions of this group were the same as those in the mutated mtDNAgroup, except that the cell lines were Lu02 (cell lines with normalmtDNA).

TABLE 1 Target region of mtDNA/ Annealing temperature Primers (5′→3′)Checking L3085-H3415 L3085: taatccaggtcg step of an (332 bps)/ gtttctextraction 58° C. (SEQ ID NO: 2) process on H3415: tatgttgatgcg mtDNAtttccg (SEQ ID NO: 3) Detection L5604-H5863 L5604: cactctgcatca process(279 bps)/ actgaacg 58° C. (SEQ ID NO: 4) H5863: agtccaatgctt cactcagc(SEQ ID NO: 5)

FIG. 4 is a result of a checking process of an extraction process onmtDNA (i.e. the step shown in FIG. 1(d)). This result indicates that themtDNA indeed can be extracted from the sample and the PMT system candetect the signals of the fluorescent reagent labeled on the mtDNA, whenthe system of Embodiment 1 was used. Conversely, if the sample was waterand did not contain mtDNA, the PMT system almost cannot detect anysignals.

FIG. 5 and FIG. 6 are detection results of normal mtDNA group andmutated mtDNA group according to the present example. As shown in FIG.5, the used ApaI cannot recognize mtDNA without mutations (i.e. normalmtDNA), so no fluorescent signal shift was found between the detectiongroup in the detecting-sample chamber and the comparison group in thecomparison sample chamber after several cycles of DNA amplifications.However, as shown in FIG. 6, the used ApaI can recognize and digestpoint-mutated mtDNA, so there a fluorescent signal shift d₁ was foundbetween the detection group in the detecting-sample chamber and thecomparison group in the comparison sample chamber after several cyclesof DNA amplifications.

COMPARATIVE EXAMPLE 1

The purpose of the present comparative example is to confirm that themethod, the device and the system for detecting mtDNA alterations ofEmbodiment 1 can accomplish similar effect as those generally used inthe art. In the present comparative example, a conventional real-timePCR machine was used, in which the reaction temperature, the sample, thesequences of the primer pair including the mutated mtDNA group and thenormal mtDNA group, and the restriction enzyme were the same as thoseused in Example 1. The results are shown in FIG. 7 and FIG. 8.

As show in FIG. 7, the used ApaI cannot recognize mtDNA withoutmutations (i.e. normal mtDNA), so no fluorescent signal shift was foundbetween the comparison group in the comparison sample chamber (withoutadding the restriction enzyme) and the detection group in thedetecting-sample chamber (with adding the restriction enzyme) afterseveral cycles of DNA amplifications. However, as shown in FIG. 8, theused ApaI can recognize and digest point-mutated mtDNA, so in that casea fluorescent signal shift d₂ was found between the comparison group inthe comparison sample chamber and the detection group in thedetecting-sample chamber after several cycles of DNA amplifications. ΔRn in the figure shows the significant fluorescent signals detected bythe analysis device.

EXAMPLE 2

The device, the system and the method for detecting mtDNA alterationsaccording to Embodiment 1 were used in the present example to detectmtDNA with 0, 30, 60, 90, 100% mutation degree. The mutation degreemeans the ratio of mutated mtDNA in total amount of extracted mtDNA inthe sample.

COMPARATIVE EXAMPLE 2

The real-time PCR machine of Comparative Example 1 was used in thepresent comparative example to detect mtDNA with 0, 30, 60, 90, 100%mutation degree.

As shown in FIG. 9, the mutation degree is directly proportional to thedetected signals. The result detected by the device, the system and themethod for detecting mtDNA alterations according to Embodiment 1 andthat detected by the conventional real-time PCR machine are similar. ΔCt is a differential value from the increasing curve of PCR detected inthe comparison sample chamber and the detecting-sample chamber in eachcycle.

The results of Examples 1-2 and Comparative Examples 1-2 will show thatthe method, the device and the system for detection mtDNA alterations ofEmbodiment 1 can incur similar effect to the conventional device.

In conclusion, the method, the device and the system for detection mtDNAalterations of the present invention indeed can work to detect mtDNAalterations. More particularly, the device provided by the presentinvention can perform cell lysis, enzyme digestion and optical detectionon a single device, so that complex treatment and detection can besimplified. In addition, the device of the present invention is a cheap,micro-sized and disposable device, so detection on mtDNA alterations canbe performed rapidly and easily accessible to the public. Hence, thedevice of the present invention can further be applied in clinicalsettings to determine whether a subject is at risk of contractingdiseases related to mitochondria gene alterations.

Although the present invention has been explained in relation to itspreferred embodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the invention as hereinafter claimed.

What is claimed is:
 1. A method for detecting mitochondria genealterations in a microfluidic device, comprising the following steps:(A) providing a microfluidic device, comprising: (i) a purification unitcomprising a first reaction chamber and a separation element storagechamber, wherein the separation element storage chamber connects to thefirst reaction chamber through a first pump, the separation elementstorage chamber comprises a separation element, and the separationelement is modified with a DNA fragment capable of recognizingmitochondrial genes; and (ii) a detection unit disposed under thepurification unit, wherein the detection unit comprises a secondreaction chamber, a detecting sample chamber, and a comparison chamber,the second reaction chamber connects to the first reaction chamber, andthe detecting sample chamber and the comparison chamber connect to thesecond reaction chamber through a second pump; (B) providing a samplecontaining mitochondria genes; (C) placing the sample into the firstreaction chamber, and introducing the separation element into the firstreaction chamber using the first pump, to separate the mitochondriagenes from the sample through a binding between the DNA fragment of theseparation element and the mitochondria genes in the sample, so as toobtain a detecting sample containing the mitochondria genes; (D)introducing a first portion of the detecting sample into the detectingsample chamber and a second portion of the detecting sample into thecomparison chamber using the second pump; (E) adding an amplificationsolution into both the detecting sample chamber and the comparisonchamber and further adding a restriction enzyme into the detectingsample chamber, wherein the amplification solution comprises a labelingreagent and a primer pair, and the restriction enzyme includes ApaI fordigesting a target gene region of the mitochondria genes; (F) performinga DNA amplified reaction in both the detecting sample chamber and thecomparison chamber, wherein the primer pair is used to amplify thetarget gene region of the mitochondria genes; (G) determining therelative amount of amplified DNA by measuring a signal from the labelingreagent in the detecting sample chamber and the comparison chamber; and(H) comparing the difference between the signals of the detecting samplechamber and the comparison chamber; wherein when the amount of signal inthe detecting sample chamber is less than that in the comparisonchamber, it indicates that a mitochondria gene alteration is present inthe sample.
 2. The method of claim 1, wherein step (D) further comprisesobtaining a third portion of the detecting sample and confirming thatmitochondria genes are present in the detecting sample by detectingmitochondria genes in the third portion.
 3. The method of claim 2,wherein the mitochondria genes are detected through a polymerase chainreaction (PCR) or a real-time polymerase chain reaction (real-time PCR).4. The method of claim 3, wherein a primer pair used in the PCR or thereal-time PCR pairs with a D-loop region of the mitochondria genes. 5.The method of claim 1, wherein the labeling reagent used in step (E) isa fluorescent dye.
 6. The method of claim 1, wherein the DNA amplifiedreaction used in step (F) is a PCR or a real-time PCR.
 7. The method ofclaim 1, wherein the separation element is a magnetic bead or a polymerbead.